In bioscience research, medical treatment, drug development and similar fields, it has become increasingly important to examine biological samples to comprehensively identify various substances, such as proteins, peptides, nucleic acids and sugar chains. In particular, when aimed at proteins or peptides, such a comprehensive analysis method is called “shotgun proteomics.” For such analyses, the combination of a chromatographic technique, such as a liquid chromatograph (LC) or capillary electrophoresis (CE), with an MSn mass spectrometer (tandem mass spectrometer) has proven itself to be a very powerful technique.
A procedure of a commonly known method for comprehensively identifying various kinds of substances in a biological sample by means of an MSn mass spectrometer is as follows:
[Step 1] Various substances contained in a sample to be analyzed are separated by an appropriate method, e.g. LC or CE. The thereby obtained eluate is preparative-fractionated to prepare a number of small amount samples. (Each of the small amount samples obtained by preparative fractionation is hereinafter called the “fractionated sample.”) The preparative fractionation of a sample should be performed in such a manner that small amount samples are collected either continuously at regular predetermined intervals of time or constantly in the same amount so that every substance in the sample will be included in one of the fractionated samples without fail.
[Step 2] For each fractionated sample, an MS1 measurement is performed to obtain an MS1 spectrum, and a peak or peaks that are likely to have originated from a substance or substances to be identified are selected on the MS1 spectrum.
[Step 3] Using the peak selected in Step 2 as the precursor ion, an MS2 measurement for the fractionated sample concerned is performed. Then, based on the result of this measurement, a database search or de novo sequencing is performed to identify a substance or substances contained in the fractionated sample.
[Step 4] If no specific substance has been identified with sufficient accuracy, an MS2 measurement using another peak on the MS1 spectrum as the precursor ion is performed, or a higher-order MSn measurement (i.e. n=3 or greater) using a specific ion observed on the MS2 spectrum as the precursor ion is performed. Then, a database search, de novo sequencing or similar data processing based on the result of the measurement is performed to identify a substance or substances contained in the fractionated sample.
[Step 5] The processes of Steps 2 through 4 are performed for each of the fractionated samples to comprehensively identify various substances contained in the original sample.
To identify each of the substances with high accuracy by the previously described comprehensive identification process, it is desirable that each fractionated sample should contain a small number of kinds of substances (most desirably, only one kind). To achieve this, it is necessary to shorten the period of each fractionating cycle, which significantly increases the number of cycles of fractionation. Considering that, to identify as many substances as possible within a limited length of measurement time or with a limited number of times of measurements, i.e. to improve the throughput of the comprehensive identification of one or more substances contained in a fractionated sample, it is necessary to preferentially select, as the precursor ion, one or more peaks having a higher probability of successful identification (which is hereinafter called the “identification probability”) among the peaks observed on the MS1 spectrum and perform the MSn analysis under appropriate measurement conditions.
One conventional method for selecting a precursor ion for an MS2 measurement from the peaks observed on an MS1 spectrum obtained for a given sample is to sequentially select the peaks on the spectrum in descending order of intensity (see Patent Document 1). For example, if the length of time for the MS2 measurement of one sample is limited, the system is controlled so that a predetermined number of peaks will be sequentially selected as the precursor ion in descending order of their intensities. In another commonly known method, all the peaks, without limiting the number of peaks, whose intensities are equal to or greater than a predetermined threshold are selected as precursor ions, provided that the measurement can be performed for an adequate length of time or an adequate number of times.
These methods seem to entirely rely on the assumption that using an ion having a higher peak intensity ensures a higher identification probability. Although this assumption is not qualitatively wrong, it should be noted that the peak intensity does not always correspond to the value of identification probability. For example, suppose that there are multiple peaks that can be chosen as a precursor ion. In some cases, choosing any one of these peaks will result in successful identification with high probability, while in other cases successful identification can be expected only when a specific peak among them is chosen. Quantitatively discriminating between such different situations from the peak intensity beforehand is considerably difficult. Thus, there has been no established method for quantitatively evaluate the identification probability of each peak on an MS1 spectrum beforehand, i.e. before the execution of an MS2 measurement, and this is one of the major factors which decrease the efficiency of the comprehensive identification.